Fatty acid analog enzyme substrates

ABSTRACT

Novel oxy- and thio-substituted fatty acid analog substrates of myristoylating enzymes are provided which contain an oxygen or sulfur in place of a methylene group in a carbon position from 4 to 13 in the fatty acid chain of a C 13  -C 14  fatty acid or alkyl ester thereof.

This invention was made in part with government support under Grant No.AI 27179, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This is a CONTINUATION of application Ser. No. 07/745,660, filed Aug.16, 1991 now abandoned, which is a Division of application Ser. No.07/208,192, filed Jun. 16, 1988, now abandoned, which is acontinuation-in-part of application Ser. No. 07/151,774, filed Feb. 3,1988, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to novel fatty acid analog substrates ofmyristoylating enzymes and, more particularly, to oxy- andthio-substituted fatty acid analogs which are useful in the fatty acidacylation of peptides and proteins.

Fatty acid acylation of specific eukaryotic proteins is a wellestablished process which can conveniently be divided into twocategories. On the one hand, palmitate (C₁₆) is linked to membraneproteins via ester or thioester linkage post-translationally, probablyin the Golgi apparatus.

On the other hand, it is known that myristate (C₁₄) becomes covalentlybound to soluble and membrane proteins via amide linkage early in theprotein biosynthetic pathway. In the N-myristoylated proteins,amino-terminal glycine residues are known to be the site of acylation.

A variety of viral and cellular proteins have been shown to be thusmodified by the covalent attachment of myristate linked through an amidebound to glycine at their amino termini. An example of a most thoroughlystudied myristoylated protein is the transforming protein of Roussarcoma virus, p 60^(v-src).

The myristoylation reaction can be represented as follows: ##STR1##

Further background information on the above protein fatty acid acylationcan be had by reference to the following series of articles byscientists associated with the Washington University School of Medicine:

Towler and Glaser, Biochemistry 25, 878-84 (1986);

Towler and Glaser, Proc. Natl. Acad. Sci. USA 83, 2812-2816 (1986);

Towler et al., Proc. Natl. Acad. Sci. USA 84, 2708-2712 (1987);

Towler et al., J. Biol. Chem. 262, 1030-1036 (1987); and

Towler et al., Ann. Rev. Biochem. In Press (1988).

Unique synthetic peptides having relatively short amino acid sequenceswhich are useful as substrates of myristoylating enzymes are describedin U.S. Pat. No. 4,740,588. Examples of such peptides are

Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg and

Gly-Asn-Ala-Ala-Ser-Tyr-Arg-Arg.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel fatty acid analogsubstrates for myristoylating enzymes are provided. These novelcompounds are oxy- and thio-substituted fatty acid analogs which areuseful in the fatty acid acylation of proteins. They contain an oxygenor sulfur in place of a methylene (--CH₂ --) group in a carbon positionfrom 4 to 13 in the fatty acid chain of a C₁₃ -C₁₄ fatty acid or alkylester thereof. The carboxyl carbon atom is defined herein as number 1based on conventional nomenclature. Preferred alkyl esters of the fattyacid analogs have from 1 to 6 carbon atoms in the alkyl group.

These novel substrate compounds are useful for studying the regulationof enzyme action in fatty acid acylation and the role ofN-myristoylation in protein function. They can serve as syntheticsubstrates for the N-myristoylating enzymes in sources such as yeasts,wheat germ lysates and mammmalian cells. These novel compounds differ inhydrophobicity from myristic acid while maintaining approximately thesame chain length. Thus, when incorporated into myristoylproteins, theyshould alter the acylprotein's subsequent interactions with membranes orwith hydrophobic proteins. They also have potential use as antiviral andantineoplastic agents.

Illustrative examples of the novel oxy- and thio-substituted fatty acidanalog substrate compounds of this invention are:

A. 11-(Ethylthio)undecanoic acid

CH₃ CH₂ S(CH₂)₁₀ COOH

B. 11-(Ethoxy)undecanoic acid

CH₃ CH₂ O(CH₂)₁₀ COOH

C. 5-(Octylthio)pentanoic acid

CH₃ (CH₂)₇ S(CH₂)₄ COOH

D. 11-(Methoxy)undecanoic acid

CH₃ O(CH₂)₁₀ COOH

E. 12-(Methoxy)dodecanoic acid

CH₃ O(CH₂)₁₁ COOH

F. 5-(octyloxy)pentanoic acid

CH₃ (CH₂)₇ O(CH₂)₄ COOH

G. 10-(Propylthio)decanoic acid

CH₃ (CH)₂ S(CH₂)₉ COOH

H. 10-(Propoxy)decanoic acid

CH₃ (CH₂)₂ O(CH₂)₉ COOH

I. 11-(1-Butoxy)undecanoic acid

CH₃ (CH₂)₃ O(CH₂)₁₀ COOH

J. 10-(2-Propynoxy)decanoic acid

HC.tbd.CCH₂ O(CH₂)₉ COOH

Alternate nomenclature can be used for the above oxy- andthio-substituted fatty acid analog substrate compounds. For example,compound A can be named 12-thiamyristic acid; compound B can be named12-oxymyristic acid; and compound J can be named 13-yne-11-oxy-myristicacid.

In a preferred embodiment of the invention the oxy- and thio-substitutedfatty acid analog substrate compounds are based on saturated C₁₃ -C₁₄fatty acids as exemplified by compounds A to H, above.

Compound I, which is a fatty acid analog based on a C₁₆ saturated fattyacid, is less effective than the analogs based on C₁₃ -C₁₄ fatty acids.

In still another embodiment, illustrated by compound J, above, the fattyacid analog is based on an ω-unsaturated C₁₄ fatty acid. It is believedthat results such as obtained with the latter compound also can beachieved with fatty acid analogs based on Δ⁹,10 cis and Δ⁹,10 transunsaturated fatty acids, e.g., 12-thiamyristoleic acid and12-oxymyristelaidic acid.

The preparation of the oxy- and thio-substituted fatty acid analogsubstrate compounds can be carried out by methods analogous to thepreparation of mixed ethers by the Williamson synthesis. Thus, anappropriate ω-bromo carboxylic acid can be reacted with an alcoholate oran alkyl thiol to produce, respectively, the oxy-substituted fatty acidether or the thio-substituted fatty acid ether.

In particular, the compounds of the invention can be produced by methodsanalogous to the synthesis of heteroatom-substituted analogs of stearicacid as described by Pascal and Ziering, J. Lipid Res. 27, 221-224(1986). Using these methods, the sulfur-containing analogs can beprepared by the condensation of appropriate alkyl thiols and ω-bromocarboxylic acids in the presence of alcoholic base. This can beillustrated by the preparation of compound A, above, as follows:##STR2##

Similarly, the oxygen-containing analogs can be prepared by the reactionof the ω-bromo acids with alcoholic base. This can be illustrated by thepreparation of compound E, above, as follows:

    Br(CH.sub.2).sub.11 COOH+CH.sub.3 OH+KOH→CH.sub.3 O(CH.sub.2).sub.11 COOH+KBr+H.sub.2 O

Other oxy- and thio-substituted fatty acid analog substrate compounds ofthe invention can be prepared by similar such methods by selectingappropriate alkyl and fatty acid chain lengths in the reactant compoundsto produce the desired products. Both of the foregoing type reactionsare carried out in organic solvent medium at refluxing temperaturesuntil the desired reaction is essentially complete.

Although specific methods of preparation of the novel fatty acid analogsare described herein, it will be understood that the novel compounds ofthis invention are not limited to any specific method of preparation.

In a typical compound of this invention, namely 11-(ethylthio)undecanoicacid, it has been found that introduction of the thioether moiety intothe fatty acid chain unexpectedly and surprisingly decreases itshydrophobicity and the hydrophobicity of the respective acyl peptidesand fatty acyl proteins, yet leaves intact its ability to act as asubstrate for the enzyme myristoyl CoA: protein N-myristoyl transferase(NMT). Purification and use of this enzyme are described, for example,by Towler et al., Proc. Natl. Acad. Sci. USA 84, 2708-2712 (1987); J.Biol. Chem. 262, 1030-1036 (1987).

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following detailed description taken in conjunctionwith the accompanying drawings in which:

FIG. 1 is a graphical representation which shows a comparison of thekinetic characteristics of myristic acid and 11-(ethylthio)undecanoicacid with wheat germ NMT. Myristoyl CoA and fatty acid analog CoA weregenerated by incubation of the fatty acid or analog with CoA, ATP andCoA ligase. This reaction mixture was added to wheat germ NMT andGly-Asn-Ala-Ala-Ser- ¹²⁵ I!Tyr-Arg-Arg in the presence of 0.03% Triton®X-100. Reaction products were characterized by reverse phase HPLC andgamma counting.

FIG. 2 is a graphical representation which shows the C-18 reverse phaseHPLC elution profiles of myristoyl-GNAASYRR ¹²⁵ I! and11-(ethylthio)undecanoyl-GNAASYRR ¹²⁵ I!. The fatty acid analogacyl-peptide eluted 7 minutes earlier than the corresponding myristoylpeptide. The free peptide elutes in the void volume (3-5 minutes).

The invention is illustrated in greater detail in the following Examples1 to 15 by the synthesis and testing of representative compounds of theinvention as myristoylating enzyme substrates. Accordingly, theinvention is first illustrated in Examples 1 to 5 by the chemicalsynthesis of a sulfur-containing analog of myristic acid, namely11-(ethylthio)undecanoic acid, CH₃ CH₂ --S--(CH₂)₁₀ COOH, which is thentested in vitro as a wheat germ NMT substrate. Wheat germ NMT is aprotein similar to yeast NMT. The purity and chemical identity of thesynthesized compound were examined using ¹ H and ¹³ C NMR as well asmass spectroscopy. The assay that was used to characterize the substratespecificity of wheat germ NMT measures the rate of attachment of aradiolabelled fatty acid to an unlabelled peptide. The fatty acid analogsynthesized was nonradioactive. The peptideGly-Asn-Ala-Ala-Ser-Tyr-Arg-Arg was labelled with Na(¹²⁵ I! in thepresence of iodogen. The reaction was chased with cold NaI to create apeptide population uniformly iodinated on Tyr⁶. Initial testsdemonstrated that the fatty acid analog did not significantly alter thekinetic characteristics of the peptide substrate: the K_(m) =3 μM withmyristic acid while the K_(m) =7 μM with 11-(ethylthio)-undecanoic acid.The apparent maximal velocity is 2.3 times higher with11-(ethylthio)-undecanoic acid than with myristic acid. The iodinatedpeptide was used at saturating concentrations for all further tests (40μM).

The CoA (Coenzyme A) ester of the sulfur-containing analog appeared tobe at least as good a substrate for wheat germ NMT as myristoyl CoAitself. FIG. 1 shows representative data from one of three testscomparing the kinetic characteristics of myristate and the sulfurcontaining analog with wheat germ NMT. The K_(m) for the analog is 1.5times higher than for myristate, while the V_(max) for the analog is 3.5times higher. This difference in K_(m) and V_(max) between analog andmyristate was noted in each test. The in vitro assay for NMT activityrequires the enzymatic generation of fatty acyl CoA. Since thePseudomonas acyl CoA ligase is quite nonspecific Shimizo et al., Anal.Biochem. 107, 193-198 (1980)! and is used in excess, it should equallywell convert myristic acid and 11-(ethylthio)undecanoic acid to thecorresponding CoA esters. It is, therefore, believed that the datarepresent the relative catalytic efficiency of fatty acyl CoA and analogCoA for wheat germ NMT.

Similar results were obtained using an 11,000-fold purified preparationof yeast NMT Towler et al., Proc. Natl. Acad. Sci. USA 84, 2708-2712(1987)!. The K_(m) and V_(max) for the analog was indistinguishable fromthat of the C14:0 fatty acid.

11-(ethylthio)undecanoic acid has another interesting characteristicwhich is believed to be useful for studying the biological role ofprotein N-myristoylation--namely its hydrophobicity is significantlydifferent from that of myristic acid. This difference was evident whenthe HPLC elution characteristics of fatty acyl peptides were examined.The elution time of a particular acyl-peptide depends strongly on thepeptide sequence as well as on the acyl chain attached to that peptide.For example, the elution times of fatty acyl derivatives of GNAAAARR are(in minutes) decanoic acid, 9; dodecanoic acid, 18; myristic acid, 24;and palmitic acid, 30. Elution times for fatty acyl derivatives of GNAAS¹²⁵ I!YRR are 23 and 28 minutes for C12 and C14 fatty acids,respectively (FIG. 2). 11-(ethylthio)undecanoyl-GNAAS ¹²⁵ I!YRR elutedseven minutes earlier than the corresponding myristoyl-peptide (FIG. 2).This suggests that the substitution of a sulfur for a carbon in thebackbone of the fatty acid chain (at least at position 12) has an effectsimilar to shortening the chain length of the fatty acid by two carbons.Such a change could alter the biological activity of myristoyl proteins.

EXAMPLE 1 Synthesis of 1l-(ethylthio)undecanoic Acid

11-Bromoundecanoic acid (1 g, 3.77 mmol, Aldrich) was added to asolution of ethanethiol (0.279 mL, 3.77 mmol, Aldrich) and potassiumhydroxide (0.486 g, 8.66 mmol) in absolute ethanol (40 mL) and refluxedfor 5 hr under a nitrogen atmosphere. After cooling and acidificationwith HCl, solvent was removed under reduced pressure to give a whitesolid. The solid was dissolved in ethyl acetate and extracted withwater. The organic phase was dried over sodium sulfate, filtered and thesolvent removed under reduced pressure. The product was purified bysilica column chromatography using increasing concentrations of ethylacetate in hexane for elution. The product eluted at 25% ethylacetate/hexane. Solvent was removed under reduced pressure to yield11-(ethylthio)-undecanoic acid (76 mg, 8%), mp 58°-61° C.; ¹ H NMR (300MHz, CDCl₃) δ 1.24 (t, 3H, J=7.4, CH₃), 1.20-1.40 (bm, 12H, methyleneenvelope), 1.48-1.67 (bm, 4H, S--CH₂ --CH₂ COO--CH₂ --CH₂), 2.33 (t, 2H,J=7.5, CH₂ --COOH), 2.49 (t, 2H, J=7.4, S--CH₂ --CH₂), 2.51 (q, 2H,J=7.4, S--CH₂ --CH₃), 10.5 (br, 1H, COOH); ¹³ C NMR (75.4 MHz, CDCl₃) δ14.88, 24.70, 25.98, 28.97, 29.08, 29.24*, 29.38, 29.48, 29.69, 31.73,34.11, 180.05; MS, m/z 246 (M⁺, 50), 217 (COOH(CH₂)₁₀ S⁺, 7), 199 (100),181 (7), 167 (7), 149 (6), 117 (7), 101 (9), 97 (9), 87 (14), 83 (18),75 (54), 69 (29), 62 (18), 55 (37).

EXAMPLE 2 Preparation of Gly-Asn-Ala-Ala-Ser- ¹²⁵ I!-Tyr-Arg-Arg

In a typical test, 100 μL of a 2 mg/ml stock (approximately 2 mM) of thepeptide Gly-Asn-Ala-Ala-Ser-Tyr-Arg-Arg, was adjusted to pH 7.4 withNaOH and diluted to 0.5 mL with PBS (30 mM sodium phosphate, pH 7.5, 150mM NaCl). This solution was added to an iodogen (Pierce) coatedpolypropylene tube and incubated with carrier free Na¹²⁵ I (400 μCi) for15 minutes at room temperature. Cold NaI was then added to a finalconcentration of 2 mM and incubation was allowed to proceed for another15 minutes. The reaction mixture was loaded onto a disposable octadecyl(C₁₈) column (Baker) which had been pre-equilibrated with 0.05%trifloroacetic acid in water. Na¹²⁵ I was removed by washing the columnwith 10 volumes of 0.05% trifloroacetic acid/water. Peptide was theneluted with 25% acetonitrile/0.05% trifloroacetic/water, and the solventremoved under a stream of nitrogen. The iodinated peptide wasresuspended in water at a final concentration of 2 mg/ml. Peptidespecific activity was 100,000 cpm/nmol.

EXAMPLE 3 Partial Purification of NMT from Wheat Germ

Procedures developed earlier by Towler et al., J. Biol. Chem. 262,1030-1036 (1987), for yeast NMT were employed for wheat germ NMTpurification with slight modifications. All steps were carried out at 4°C. unless otherwise indicated. Protein was quantitated by the method ofBradford, Anal. Biochem. 72, 248-254 (1986). NMT activity was assayed asdescribed by Towler and Glaser, Biochemistry 25, 878-884 (1976), using asynthetic peptide Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys, derived from theN-terminal sequence of p60^(v-src). This peptide was used because of itsgreater stability in crude enzyme fractions compared to other peptidesubstrates. Seven hundred and fifty mL of buffer A (20 mMN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.6, 100 mMpotassium acetate, 1 mM magnesium acetate, 2 mM calcium chloride, 1 mMDTT) prechilled to 4° C. was added to 300 g of untoasted wheat germ. Theslurry was homogenized in a Waring blender for 15 seconds. The samplewas then centrifuged at 27,500×g for 20 minutes. The resultingsupernatant was filtered through cheesecloth and concentrated overnightin an Amicon concentrator with a YM30 membrane.N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.6) was addedto a final concentration of 0.1 M. Sequential ammonium sulfatefractionation was carried out as described by Towler et al., J. Biol.Chem. 262, 1030-1036 (1987). After resuspending the 25-50% fraction inbuffer B (50 mM Tris HC1, pH 7.4, 1 mM DTT, 0.1 mM EGTA, 1 μg/mL each ofthe peptide protease inhibitors aprotinin, soybean trypsin inhibitor,leupeptin and pepstatin) the sample was dialyzed against three changesof buffer B (4 L per exchange). The sample was then diluted 4-fold andcentrifuged for 10 minutes at 11,750×g. An aliquot of the supernatant(90 ml of 420 ml) was loaded (at 200 mL/hr) onto a 5 ×23-cmDEAE-Sepharose® CL-6B column (Pharmacia P-L Biochemicals)preequilibrated with buffer C (20 mM Tris HCl, pH 8, 1 mM DTT). Thecolumn was successively washed with one column volume of buffer C plus0, 50, 100, or 200 mM NaCl. Material with the highest specific activityeluted in the 100 mM NaCl wash (fractions 59-72) and was used forfurther purification. After concentrating to ˜40 mL with a YN30membrane, this fraction was dialyzed (2×2 L) against buffer D (10 mMTris HC1, pH 7.4, 1 mM DTT, 0.1 mM EGTA), plus the peptide proteaseinhibitors listed above. It was subsequently added to a 5 mL slurry ofType V AG-CoA (CoA-agarose affinity matrix; Pharmacia P-L Biochemicals)pre-equilibrated with buffer D plus protease inhibitors. After 5 hrs ofcontinuous mixing, the suspension was poured into a 2.5 cm high×1.5 cmdiameter column and washed with three column volumes of buffer D. Thiswas followed by sequential step elutions with 100, 200 or 500 mM KCl inbuffer D (3 column volumes each). NMT activity eluted in the 200 and 500mM KCl washes. These fractions were combined, dialyzed against buffer E(10 mM potassium phosphate, pH 7.4, 1 mM DTT) and loaded onto a Bio-GelHTP column (5 g of hydroxylapatite; Bio-Rad) as described by Towler etal., J. Biol. Chem. 262, 1030-1060 (1987). NMT activity was eluted with200 mM potassium phosphate. This fraction was concentrated and dialyzedwith buffer D using Centricon-30 microconcentrators (Amicon). Partiallypurified NMT was stored at 4° C. as a 77 mg/mL protein solution andused, after appropriate dilution, for all further characterization.(DTT=DL-dithiothreitol;EGTA=ethyleneglycol-bis-(β-aminoethylether)--N,N,N',N'-tetraaceticacid.)

The purification results are set forth in the following table.

                  TABLE 1    ______________________________________    Partial Purification of Wheat Germ NMT                     Protein    Specific                                        Fold            Volume.sup.a                     Concentration                                Activity                                        Puri- %    Fraction            (mL)     (mg/mL)    (units.sup.b /mg)                                        cation                                              Yield    ______________________________________    Initial 60       126        0.67    1     100    Extract    25-50%  90       32         4.2     6.3   239    Ammonium    Sulfate    Fraction    DEAE    39       8          17.5    26    108    Column    CoA Column            21       0.71       293     437    86    Hydroxy-            0.087    77         323     480    43    apatite    Column    ______________________________________     .sup.a Volumes of the initial extract and ammonium sulfate fraction liste     in this Table are 20% of the actual volumes used at these stages of     purification since only 20% of the sample was used for subsequent     purification steps.     .sup.b One unit of activity is defined as 1 pmol acylpeptide formed/min.

EXAMPLE 4 Characterization of Partially Purified Wheat Germ NMT

Assays for NMT activity were performed using a procedure detailedpreviously by Towler and Glaser, Proc. Natl. Acad. Sci. USA 83,2812-2816 (1986). Briefly, the in vitro assay measures the transfer ofradiolabelled fatty acid from acyl CoA to a synthetic peptide substrate.The acylpeptide product is identified by reverse phase HPLC. Time andenzyme dependence were evaluated by varying the reaction time or theenzyme concentration. A 10 minute reaction time was found to be in thelinear range for product formation (data not shown). Fatty acidspecificity was examined essentially as described by Towler et al., J.Biol. Chem. 262, 1030-1036 (1987), except that the peptideGly-Ser-Ser-Lys-Ser-Lys-Pro-Lys rather thanGly-Asn-Ala-Ala-Ala-Ala-Arg-Arg was used to compare palmitate andmyristate utilization by wheat germ NMT. This was necessary because ofthe presence of a large, peptide-independent peak which co-eluted with apalmitoyl-Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg standard. ³ H!Palmitate and ³H!myristate were used in this comparison. ¹⁴ C!fatty acids were used tocompare C-10, C-12 and C-14 chain lengths. Fatty acyl CoA concentrationsin these assays were determined by a modification of the method ofHosaka et al., Methods Enzymol. 71, 325-333 (1981).

EXAMPLE 5 Characterization of the Fatty Acid Analog

Gly-Asn-Ala-Ala-Ser- ¹²⁵ I!Tyr-Arg-Arg as prepared in Example 2, above,was tested in an in vitro myristoylation system which was modified fromthat used with radiolabelled fatty acids. To determine the K_(m) of thepeptide with myristic acid and the thioether analog as substrates, coldmyristate and 11-(ethylthio)undecanoic acid were used in the acyl CoAgenerating system Towler et al., J. Biol. Chem. 262, 1030-1036 (1987);Towler and Glaser, Proc. Natl. Acad. Sci. USA 83, 2812-2816 (1986)! at afinal concentration of 5 μM. 20-fold more wheat germ NMT (480-foldpurified as in Example 3, above) was used for these assays than forassays which employed ³ H! myristate. This amount of NMT was found to bein the linear range of enzyme concentration (data not shown). Doublereciprocal plots were generated for the iodinated peptide with bothmyristic acid and the sulfur analog by altering the peptideconcentration in the presence of a constant amount of fatty acid oranalog. For determination of the Km of 11-(ethylthio)undecanoic acid andcold myristic as their CoA esters, saturating concentrations ofiodinated peptide (40 μM) were used and the level of fatty acid oranalog was varied. The assays contained 0.03% Triton X-100 to maintainthe solubility of the fatty acids. The results are shown in FIG. 1.

Other illustrative examples of the oxy- and thio-substituted fatty acidanalog substrate compounds of the invention were synthesized in Examples6 to 14 and tested in Example 15 as substrates for the yeast NMT. Acomparison of the kinetic characteristics of these analog substratesusing myristic acid as a standard is set forth in the Table 2.

For these Examples, each analog synthesized is characterized by ¹ H and13C NMR as well as mass spectroscopy. The analogs are then characterizedkinetically as NMT substrates. Briefly, fatty acid analogs are convertedto their CoA esters with Pseudomonas CoA ligase and incubated with theiodinated peptide GNAAS ¹²⁵ I!YRR and a source of NMT. Peptide K_(m) andV_(m) are first determined by varying peptide concentration with fattyacid analog at 15 μM (generally a saturating concentration). Theradiolabelled peptide is then used at concentrations which will lead to50% enzyme saturation (i.e., at its K_(m)) for the determination offatty acid analog kinetic characteristics. Representative analogs withsulfur or oxygen in the backbone of the fatty acid chain serve as goodsubstrates for NMT in vitro and will compete for incorporation of ³ Hlabelled myri-state into yeast proteins in vivo. In addition, theseanalogs differ markedly in hydrophobicity from myri-state as measuredboth by C₁₈ reverse phase HPLC elution profiles and by 2-octanol/waterpartition coefficients. The analog CH₃ O (CH₂)₁₁ COOH, for example,partitions into water 20 fold better than myristate. It is believed thatthese analogs will be incorporated into mammalian acylproteins in vivoand that their incorporation into these proteins will drastically alterprotein processing or targeting. It is further believed that theincorporation of fatty acid analogs of both myristate and palmitate intoester linked and phosphatidyl inositol glycan linked acylproteins willalso dramatically alter their properties. Since many viral proteins andoncogenes are acylated, these compounds represent a new class ofpotential antiviral and antineoplastic agents.

EXAMPLE 6 Synthesis of 11-(ethoxy)undecanoic acid

11-bromoundecanoic acid (2.25 g, 8.47 mmol) was added to a solution ofpotassium hydroxide (2.15 g, 38.3 mmol) in absolute ethanol (20 mL) andrefluxed for 7 hrs. After cooling and acidification with HCl, solventwas removed under reduced pressure to give a white solid. The sample wasdissolved in ethyl acetate and extracted with water. The organic phasewas dried over sodium sulfate, and the solvent was removed under reducedpressure. The product was purified by silica column chromatography in 1%diethyl ether/0.3% formic acid/methylene chloride. Solvent was removedunder reduced pressure to yield 11-(ethoxy)undecanoic acid (680 mg,35%): mp 44°-45.5° C.; ¹ H NMR (300 MHz, CDCl₃) δ 1.20 (t, 3H, J=7.0,CH₃), 1.24-1.40 (bm, 12H, methylene envelope), 1.52-1.68 (bm, 4H, O--CH₂--CH₂ ; CH₂ --CH₂ --COOH), 2.34 (t, 2H, J=7.5, CH₂ --COOH), 3.41 (t, 2H,J=6.8, 0--CH₂ --CH₂), 3.48 (g, 2H, J=7.0, 0--CH₂ --CH₃), 10.25 (br, 1H,COOH); ¹³ C NMR (75.4 MHz, CDCL₃) δ 15.27, 24.75, 26.23, 29.11, 29.26,29.40, 29.55*, 29.80, 34.12, 66.06, 70.76, 179.71; m/z 231 (M+H⁺).

EXAMPLE 7 A. Synthesis of Methyl 6-thiotetradecanoate

n-Butyllithium (8.3 mL, 22.1 mmol) was added dropwise to a solution ofoctanethiol (1; 2.9 g, 19.8 mmol) in dry THF (198 mL) at 0° C. Afterstirring at 0° C. for 30 min, a solution of methyl 5-bromopentanoate (2;4.2 g, 21.6 mmol) in dry THF (43 mL) was added dropwise and theresulting heterogeneous mixture was stirred overnight at roomtemperature. The mixture was concentrated and the residue waspartitioned between ether and saturated NH₄ Cl. After extracting theaqueous layer a second time with ether, the combined organic extractswere washed with brine, dried (MgSO₄) and concentrated. Purification byreduced pressure distillation (140°-145° C. at 2 mmol) afforded 4.4 g(86%) of the title product. ¹ H-NMR data, δ 3.63 (s, 3H, OCH₃); 2.48 (q,4H, J=6.8 Hz, CH₂ --S--CH₂); 2.30 (t, J-7.0 Hz, CH₂ CO₂ CH₃); 1.78-1.48(bm, 6H, CH₂ 's beta to thio and ester moieties); 1.43-1.15 (bs, 10H,CH₂ 's); 0.85 (t, J=6.6 Hz, CH3); ¹³ C-NMR data: δ 173.7, 51.4, 33.5,32.1, 31.7, 31.6, 29.6, 29.1 (2), 29.0, 28.8, 24.1, 22.5, 14.0.

B. Synthesis of 5-(Octylthio)pentanoic acid

NaOH (1.24 g, 31.0 mmol) was added to a solution of methyl6-thiotetradecanoate (4.25 g, 16.3 mmol) in dry methanol (55 mL) and theresulting mixture brought to reflux. After 5 h the reaction was cooledto room temperature, diluted with 100 ml of water and acidified with 1 MHCl to a pH of 3. This acidified solution was extracted with ether (2X)and the combined organic extracts were washed in water (2X), brine (2X),dried (MgSO₄) and concentrated. Column chromatography (ethylacetate-pentane, 1:9) of the residue afforded 1.4 g, 35% of product. ¹H-NMR data, δ 2.46 (q, 4H, J=7.6 Hz, CH₂ SCH₂); 2.33 (t, 2H, J=7.2 Hz,CH₂ CO₂ H); 1.75-1.45 (bm, 6H, CH₂ 's beta to thio and acid moieties);1.38-1.15 (bs, 1OH, CH₂ 's); 0.83 (t, 3H, J=6.6 Hz, CH₃); ¹³ C-NMR data,179.4, 33.5, 32.1, 31.8, 31.6, 29.7, 29.1 (2), 28.9 (2), 23.8, 22.6,14.0; m/z (E1): 246, 145 (100%), 115, 101, 88, 69.

EXAMPLE 8 Synthesis of 11-(methoxy)undecanoic acid

11bromoundecanoic acid (10.0 g, 37.7 mmol) was added to a solution ofpotassium hydroxide (24.3 g, 433 mmol) in methanol (280 mL) and refluxedfor 5 hrs. After cooling and acidification with HCl, solvent was removedunder reduced pressure. The sample was dissolved in ethyl acetate andextracted with water. The organic phase was dried over sodium sulfate,and the solvent removed under reduced pressure. The product was purifiedby silica column chromatography using increasing concentrations of ethylacetate in hexanes for elution. The product eluted in 25% ethyl acetatein hexanes. Solvent was removed under reduced pressure to give11-(methoxy)undecanoic acid (200 mg, 2.5%): mp 31°-32° C.; ¹ H NMR (300MHz, CDCl₃) δ 1.1-1.3 (bm, 12H, methylene envelope), 1.45-1.63 (bm, 4H,O--CH₂ --CH₂, CH₂ --CH₂ --COOH), 2.34 (t, 2H, J=7.3, CH₂ --COOH), 3.45(s, 3H, CH₃), 3.50 (t, 2H, J=6.8, 0--CH₂), 10.70 (br, COOH); ¹³ C NMR(75.4 MHz, CDCl₃) δ 24.64, 26.00, 29.00, 29.16, 29.29, 29.40, 34.00,58.23, 72.81, 179.17; m/z 216 (M⁺).

EXAMPLE 9 Synthesis of 12-(methoxy)dodecanoic acid

12-bromododecanoic acid (2.0 g, 7.16 mmol) was added to a solution ofpotassium hydroxide (1.61 g, 28.65 mmol) in methanol (30 mLs) andrefluxed for 20 hrs. After cooling and acidification with HCl, solventwas removed under reduced pressure. The sample was dissolved in ethylacetate and extracted with water. The organic phase was dried oversodium sulfate, and the solvent removed under reduced pressure. Theproduct was purified by silica column chromatography in 1% diethylether/0.3% formic acid/methylene chloride to yield12-(methoxy)dodecanoic acid (640 mg, 39%): mp 45°-47° C.; ¹ H NMR (300MHz, CDCl₃) δ 1.20-1.45 (bm, 15H, methylene envelope), 1.51-1.69 (bm,4H, 0--CH₂ --CH₂, CH₂ --CH₂ --COOH), 2.34 (t, 2H, J=7.4, CH₂ --COOH),3.34 (s, 3H, 0--CH₃), 3.38 (t, 2H, J=6.7, 0--CH₂), 10.99 (br, 1H, COOH);¹³ C NMR (75.4 MHz, CDCl₃) δ 24.75, 26.16, 29.10, 29.27, 29.38, 29.44,29.53, 34.12, 58.50, 72.97, 179.70; m/z 231 (M+H⁺).

EXAMPLE 10 Synthesis of 5-(octyloxy)pentanoic acid

5-bromopentanoic acid (2g, 11.0 mmol) was added to a solution ofpotassium hydroxide (2.48 g, 44.2 mmol) in 1-octanol (20 mL) and stirredat 97° C. for 27 hrs. After cooling, the product was extracted at pH=1with ethyl acetate and water. The organic phase was dried over sodiumsulfate and solvent was removed. 1-octanol was removed by short path(Kugelrohr) distillation (50°-70° C., 0.5 mm Hg). To ensure completecleavage of the octyl ester, the residue was stirred 3 hrs withmethanol/water/KOH (50%/50%/2.5 g, 40 mL) at 25° C. 1-octanol wasextracted into ethyl acetate at pH=12. 5-(octyloxy)pentanoic acid wasextracted from the aqueous phase into ethyl acetate after adjusting thepH to 1.5 with HCl. After drying over sodium sulfate, the solvent wasremoved at reduced pressure. Silica column chromatography in 10% ethylacetate/hexane/0.3% formic acid, yielded 5-(octyloxy)pentanoic acid (235mg, 9%): mp<30° C.; ¹ H NMR (300 MHz, CDCl₃) δ 0.88 (t, 3H, J=6.6),1.18-1.39 (bm, 10H, methylene envelope), 1.48-1.77 (bm, 6H, CH₂ CH₂ OCH₂CH₂, CH₂ CH₂ COOH), 2.39 (t, 2H, J=7.1, CH₂ COOH), 3.34-3.49 (bm, 4H,CH₂ OCH₂), 10.66 (br, 1H, COOH); ¹³ C NMR (75.4 MHz, CDCl₃) 14.18,21.59, 22.73, 26.22, 29.03, 29.32, 29.51, 29.72, 31.89, 33.85, 70.22,71.07, 179.55; m/z 231 (M+H).

EXAMPLE 11 Synthesis of 10-(propylthio)decanoic acid

10-bromodecanoic acid (1.0 g, 3.98 mmol) was added to a solution ofpotassium hydroxide (0.893 g, 15.9 mmol) in 1-propanethiol (30 mL) andmethanol (30 mL) and stirred at 69° C. for 18 hrs. The reaction wasallowed to cool to room temperature after the addition of 20 mL water.After acidification to pH=1 and extraction into ethyl acetate, theorganic phase was dried over sodium sulfate and solvent removed atreduced pressure to yield a white crystalline powder. The product waspurified by silica column chromatography in 8% ethyl acetate/hexane/0.3%formic acid and recrystallization from hexane to yield10-(propylthio)decanoic acid (210 mg, 21%), mp 42°-43.5° C.; ¹ H NMR(300 MHz, CDCl₃) δ0.96 (t, 3H, J=7.3, CH₃) 1.15-1.40 (bm, 10H, methyleneenvelope), 1.49-1.64 (bm, 6H, CH₃ CH₂ CH₂ SCH₂ CH₂, CH₂ CH₂ COOH), 2.32(t, 2H, J=7.6, CH₂ COOH), 2.40-2.55 (bm, 4H, CH₃ CH₂ CH₂ SCH₂), 10.93(Br, 1H, COOH); ¹³ C NMR (75.4 MHz, CDCL₃) δ 13.64, 23.09, 24.71, 28.96,29.07, 29.13, 29.24, 29.36, 29.77, 32.16, 34.10, 34.28, 179.98; m/z 247(M+H).

EXAMPLE 12 Synthesis of 10-(propoxy)decanoic acid

10-bromodecanoic acid (1 g, 3.98 mmol) was added to a solution ofpotassium hydroxide (0.893 g, 15.9 mmol) in n-propanol (30 mL) andstirred at 102° C. for 18 hrs. The reaction was allowed to cool to roomtemperature after the addition of 20 mL water. After acidification topH=1 and extraction into ethyl acetate, the organic phase was dried oversodium sulfate and solvent removed at reduced pressure to yield a yellowoil. The product was purified by silica column chromatography in 2%diethyl ether/methylene chloride/0.2% formic acid, and then in 7% ethylacetate/hexane/0.3% formic acid. Recrystallization from hexane at -20°C. yielded 10-(propoxy)decanoic acid (74 mg, 12%): mp <30° C.; ¹ H NMR(300 MHz, CDCl₃) δ 0.91 (t, 3H, J=7.5, CH₃), 1.18-1.40 (br, 10H,methylene envelope), 1.48-1.67 (bm, 6H, CH₃ CH₂ CH₂ OCH₂ CH₂, CH₂ CH₂COOH), 2.33 (t, 2H, J=7.4, CH₂ COOH), 3.31-3.45 (bm, 4H, CH₂ OCH₂),10.37 (br, 1H COOH); ¹³ C NMR (75.4 MHz, CDCl₃) 10.67, 22.94, 24.72,26.19, 29.09, 29.22, 29.41, 29.74, 34.11, 70.88, 72.53, 179.69; m/z 231(M+H).

EXAMPLE 13 Synthesis of 11-(1-butoxy)undecanoic acid

11-bromoundecanoic acid (2 g, 17.5 mmol) was added to a solution ofpotassium hydroxide (1.7 g, 30.2 mmol) in 1-butanol (20 mL) and thesolution was stirred at 40° C. for 5 hrs. After cooling, the reactionmixture was extracted with ethyl acetate and water at pH=2. The organicphase was then washed with saturated sodium chloride, dried over sodiumsulfate, and the solvent was removed under reduced pressure. The productwas purified over silica column chromatography in 2-10% ethylacetate/hexane/0.2% formic acid to yield 11-(1-butoxy)undecanoic acid(336 mg, 17%). mp 29°-30.5° C.; ¹ H NMR (300 MHz, CDCl₃) δ 0.84-0.96 (t,3H, J=7.3, CH₃), 1.18-1.46 (bm, 14H, methylene envelope), 1.47-1.68 (bm,6H, CH₂ CH₂ OCH₂ CH₂, CH₂ CH₂ COOH), 2.31 (t, 2H, CH₂ COOH), 3.32-3.46(bm, 4H, CH₂ OCH₂), 11.02 (br, 1H, COOH); ¹³ C NMR (75.4 MHz, CDCl₃ δ14.02, 19.43, 24.74, 26.21, 29.11, 29.28, 29.35, 29.56, 29.64, 29.76,31.85, 34.14, 70.63, 70.94, 179.82; m/z 259 (M+H).

EXAMPLE 14 Synthesis of 10-(2-propynoxy)decanoic acid

Sodium hydride (420 mg, 8.75 mmol) was added to propargyl alcohol (68mL, 1.17 mol) at 4° C. and stirred for 30 minutes at 25° C.10-Bromedecanoic acid (2 g, 7.96 mmol) was added to this mixture and thereaction was stirred at 98° C. for 48 hrs. The reaction mixture wasextracted with water and ethyl acetate at pH=0. After drying the organicphase over sodium sulfate, the solvent was removed at reduced pressure.Product was purified over silica gel chromatography in 7-10% ethylacetate/hexane/0.3% formic acid and then over a second silica gel columnin 1-2.5% ethyl acetate/benzene/0.3% formic acid to yield10-(2-propynoxy)decanoic acid (245 mg, 14%). mp <30° C.; ¹ H NMR (300MHz, CDCl₃) δ 1.25-1.42 (br, 10H, methylene envelope), 1.54-1.70 (bm,4H, OCH₂ CH₂, CH₂ CH₂ COOH), 2.35 (t, 2H, J=7.4, CH₂ COOH), 2.43 (t, 1H,J=2.3, HC=C), 3.52 (t, 2H, J=6.8, OCH₂), 4.14 (d, 2H, J=2.5, C=CCH₂ O),10.42 (br, 1H, COOH); ¹³ C NMR (75.4 MHz, CDCl₃) δ 24.68, 26.06, 29.04,29.17, 29.35*, 29.46, 34.09, 57.95, 70.22, 74.09, 79.90, 180.02; m/z 227(M+H).

EXAMPLE 15 A. Labeling and Extraction of Yeast Proteins

Saccharomyces cerevisiae strain BJ405 MATα, trpl, prbl, prcl, pep4-3;Hemmings et al., Proc. Natl. Acad. Sci. USA 78, 435-439 (1981)!(previously referred to as JR153) was grown to mid-log phase (OD_(660nm)=1.0-1.2) in YPD medium (1% yeast extract/2% Bactopeptone/2% Dextrose)at 30° C. in a rotary shaker water bath. Cells were pretreated withCerulenin at 2 μg/ml for 15 minutes and then incubated for 20-45 minuteswith unlabeled myristate, palmitate or fatty acid analog at 100 μM inthe presence of either 5 μM 9,10-³ H(N)!-myristate (22.4 Ci/mmol) or L-³⁵ S!methionine (1106 Ci/mmol) at 30 μCi/ml of culture.

Cells were cooled on ice 5 minutes, pelleted at 10,000×g, then brokenand extracted by the method of Towler et al., Proc. Natl. Acad. Sci. USA83, 2812-2816 (1986). Total protein synthesis was assayed bytrichloroacetic acid (TCA) precipitation. Proteins were separated bySDS-12% polyacrylamide gel electrophoresis, and ³ H!fatty acidincorporation into cellular acyl-proteins was assayed by autoradiographyfollowed by laser densitometry.

B. Results

Incubation of cells with the 12-(methoxy)dodecanoic acid analog resultedin a 35-60% reduction in the incorporation of ³ H!myristate into a knownyeast myristoylprotein of M_(r) 20 kDa Towler et al, PNAS 83, 2812-2816(1986)! compared to control cells which were incubated either with ³H!myristate alone (i.e. no analog) or 100 μM palmitate. By contrast,addition of ³ H!myristate and 100 μM myristate results in a 75-80%reduction in the incorporation of ³ H!myristate into this cellularacylprotein.

The analog does not appear to be toxic to cells. No difference in thegrowth rates were observed between cells treated with 100 μM myristateand 100 μM analog. Furthermore, 100 μM myristate and 100 μM analogproduced a similar modest reduction (10-15%) in total protein synthesis(measured by the incorporation of ³⁵ S!methionine into TCA precipitableprotein) compared to control cells which were not exposed to exogenousfatty acid.

Together these results indicate that the analog can successfully enteryeast and compete with labeled myristate for incorporation into a knownmyristoylprotein.

                  TABLE 2    ______________________________________    Kinetics of Fatty Acid Analogs                          Pep-                  Elution tide   Pep- Acyl                  Time    K.sub.m                                 tide CoA   Acyl    Analog        (min.)  μM  V.sub.m                                      K.sub.m μM                                            CoA V.sub.m    ______________________________________    CH.sub.3 (CH.sub.2).sub.12 COOH*                  26      10     100% 0.6   100%    CH.sub.3 CH.sub.2 S(CH.sub.2).sub.10 COOH                  18      19      98% 1.4   130%    CH.sub.3 CH.sub.2 O(CH.sub.2).sub.10 COOH                  12      14.8    62% 1.8    64%    CH.sub.3 (CH.sub.2).sub.7 S(CH.sub.2).sub.4 COOH                  21      11.5   213% 1.5   160%    CH.sub.3 O(CH.sub.2).sub.10 COOH                  12-13   47.3   232% 6.9   163%    CH.sub.3 O(CH.sub.2).sub.11 COOH                  12-13   19.1   177% ˜2                                            ˜110%    CH.sub.3 (CH.sub.2).sub.7 O(CH.sub.2).sub.4 COOH                  16      31     335% 1.6   675%    CH.sub.3 (CH.sub.2).sub.2 S(CH.sub.2).sub.9 COOH                  18      47     245% 1.5   250%    CH.sub.3 (CH.sub.2).sub.2 O(CH.sub.2).sub.9 COOH                  11-12   34     150% 6.1   250%    CH.sub.3 (CH.sub.2).sub.3 O(CH.sub.2).sub.10 COOH                  19      42      25% 1      80%    HC.tbd.CCH.sub.2 O(CH.sub.2).sub.9 COOH                   9      28     200% 6.5   320%    ______________________________________     *Myristate control

It will be seen that the K_(m) for each of the analogs is higher thanfor myristate, while the V_(max) for the analogs is higher in mostcases.

Standard amino acid abbreviations are used to identify the sequence ofthe peptides herein as follows:

    ______________________________________    Amino Acid           Abbreviation    ______________________________________    L-Alanine            Ala or A    L-Arginine           Arg or R    L-Asparagine         Asn or N    L-Aspartic acid      Asp or D    L-Glutamine          Gln or Q    L-Glycine            Gly or G    L-Leucine            Leu or L    L-Lysine             Lys or K    L-Proline            Pro or P    L-Serine             Ser or S    L-Tyrosine           Tyr or Y    L-Valine             Val or V    ______________________________________

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. All such other examples are included withinthe scope of the appended claims.

What is claimed is:
 1. A method of acylating a peptide or proteincomprising reacting the CoA ester of an oxy- or thio-substituted fattyacid analog compound having activity as a substrate of myristoylatingenzymes selected from the group consisting of C₁₃ or C₁₄ fatty acids oralkyl esters thereof in which a methylene group normally in a carbonposition from 4 to 13 is replaced with oxygen or sulfur with saidpeptide or protein in the presence of a source of N-myristoyltransferase to thereby decrease the hydrophobicity of the resulting acylpeptide or protein compared to the corresponding myristoyl peptide orprotein while maintaining about the same chain length.